GSM7385286: 4F2A cell, DMSO, DAY2_3; Mus musculus; RNA-Seq
DOX wasaddedinD0,DMSO or CX-5461 was added in D0 for only 1 hour, the total RNA of 4F2A MEFs was harvested by TRIzol in Day2. Each group was prepared with two parallel replicates.  All the samples were sent to  BMKCloud Corporation (www.biocloud.net) by using NovaSeq 6000 for RNA sequencing. thetotalRNAwasextractedaccordingtotheinstructionmanualoftheTRlzolReagent(Lifetechnologies,California,USA).RNAconcentrationandpuritywasmeasuredusingNanoDrop2000(ThermoFisherScientific,Wilmington,DE).RNAintegritywasassessedusingtheRNANano6000AssayKitoftheAgilentBioanalyzer2100system(AgilentTechnologies,CA,USA).Atotalamountof1μgRNApersamplewasusedasinputmaterialfortheRNAsamplepreparations.SequencinglibrariesweregeneratedusingHieffNGSUltimaDual-modemRNALibraryPrepKitforIllumina(YeasenBiotechnology(Shanghai)Co.,Ltd.)followingmanufacturer'srecommendationsandindexcodeswereaddedtoattributesequencestoeachsample.Briefly,mRNAwaspurifiedfromtotalRNAusingpoly-Toligo-attachedmagneticbeads.FirststrandcDNAwassynthesizedandsecondstrandcDNAsynthesiswassubsequentlyperformed.Remainingoverhangswereconvertedintobluntendsviaexonuclease/polymeraseactivities.Afteradenylationof3'endsofDNAfragments,NEBNextAdaptorwithhairpinloopstructurewereligatedtoprepareforhybridization.ThelibraryfragmentswerepurifiedwithAMPureXPsystem(BeckmanCoulter,Beverly,USA).Then3μlUSEREnzyme(NEB,USA)wasusedwithsize-selected,adaptor-ligatedcDNAat37°Cfor15minfollowedby5minat95°CbeforePCR.ThenPCRwasperformedwithPhusionHigh-FidelityDNApolymerase,UniversalPCRprimersandIndex(X)Primer.Atlast,PCRproductswerepurified(AMPureXPsystem)andlibraryqualitywasassessedontheAgilentBioanalyzer2100system.ThelibrariesweresequencedonIlluminaNovaSeq6000platformtogenerate150bppaired-endreads,accordingtothemanufacturer'sinstructions.Therawreadswerefurtherprocessedwithabioinformaticpipelinetool,BMKCloud(www.biocloud.net)onlineplatform.Rawdata(rawreads)offastqformatwerefirstlyprocessedthroughin-houseperlscripts.Inthisstep,cleandata(cleanreads)wereobtainedbyremovingreadscontainingadapter,readscontainingploy-Nandlowqualityreadsfromrawdata.Atthesametime,Q20,Q30,GC-contentandsequenceduplicationlevelofthecleandatawerecalculated.Allthedownstreamanalyseswerebasedoncleandatawithhighquality.